Pancreatic cancer is a leading cause of cancer-related mortality in the United States, with an estimated 35,000 people dying from this disease in 2009. Activating mutations in the KRAS2 oncogene occur in >90% of pancreatic ductal adenocarcinomas (PDAC), the most common type of pancreatic cancer, and are believed to be initiating lesions in PDAC. Elevated levels of hedgehog pathway components are found in a majority of PDAC specimens and cell lines. Loss of the Smad4 transcriptional regulator occurs in approximately half of PDAC cases, and inactivation of the TGF?RII, BMPRII, and ALK4 receptor kinases that act upstream of Smad4 occurs in a smaller subset of PDAC cases. Yet, the precise roles of the hedgehog pathway and the signaling pathways activated by TGF-? family ligands in PDAC remain unclear. This application therefore proposes to use a novel mouse modeling system and complementary cell culture and in vitro studies to systematically investigate the roles of these pathways in pancreatic carcinogenesis. Prior studies have provided conflicting data on whether hedgehog ligands act in both autocrine and paracrine fashion, or only a paracrine manner, during pancreatic tumor development. Therefore, studies in Aim 1 will use novel mouse models to directly compare the ability of sonic hedgehog (Shh) and its downstream transcription factors Gli1 and Gli2 to cooperate with activated Kras during pancreatic tumorigenesis in vivo. Analyses of tumor samples and cell lines generated from the tumors induced in this Aim will be performed to determine whether Shh stimulates signaling in both the cancer cells and the reactive stroma, or only within the reactive stroma. Finally, using a novel dominant-repressor Gli3 allele, the requirement for Gli transcription activity in Kras-induced pancreatic tumorigenesis will be determined. The studies in Aim 2 will identify the relative contributions of defects in TGF-? and BMP signaling to pancreatic tumorigenesis. ShRNAs targeting Smad4, TGF?RII, or BMPRII will be delivered specifically to the pancreatic epithelium in vivo, and their ability to cooperate with activated Kras determined. Complementary experiments in cancer cell lines derived from the induced tumors will be performed to determine whether defects in TGF-( or BMP signaling alter the phenotypes of pancreatic cancer cells, and how TGF-( family ligands influence pancreatic cancer cell behavior. The studies in Aim 3 will determine whether there is cross-talk between the hedgehog and TGF-( signaling cascades in pancreatic cancer cells, and how this cross-talk influences the phenotypes of these cells. Finally, gene expression profiling will be used to identify important genes downstream of the hedgehog and TGF-( signaling cascades. The biological importance of these genes during pancreatic tumorigenesis will be validated using studies of the cancer cell lines generated in Aims 1 and 2, and the ability of these genes to cooperate with activated Kras during pancreatic tumorigenesis in vivo.